WO2012128785A1 - Méthodes et compositions pour traiter le cancer ou d'autres maladies - Google Patents

Méthodes et compositions pour traiter le cancer ou d'autres maladies Download PDF

Info

Publication number
WO2012128785A1
WO2012128785A1 PCT/US2011/051042 US2011051042W WO2012128785A1 WO 2012128785 A1 WO2012128785 A1 WO 2012128785A1 US 2011051042 W US2011051042 W US 2011051042W WO 2012128785 A1 WO2012128785 A1 WO 2012128785A1
Authority
WO
WIPO (PCT)
Prior art keywords
stat3
cpg
sirna
cells
tumor
Prior art date
Application number
PCT/US2011/051042
Other languages
English (en)
Inventor
Hua Yu
Marcin Kortylewski
Richard Jove
Piotr Marek SWIDERSKI
John J. Rossi
Original Assignee
City Of Hope
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by City Of Hope filed Critical City Of Hope
Publication of WO2012128785A1 publication Critical patent/WO2012128785A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3183—Diol linkers, e.g. glycols or propanediols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the present invention relates to methods and compositions for the treatment of diseases, including cancer, infectious diseases and autoimmune diseases.
  • the present invention also relates to methods and compositions for improving immune function. More particularly, the present invention relates to multifunctional molecules that are capable of being delivered to cells of interest for the treatment of diseases and for the improvement in immune function.
  • Signal Transducer and Activator of Transcription 3 (Stat3) is constitutively activated at high frequency (50 to 100%) in diverse cancers (Yu and Jove, 2004; Yu et al., 2007; Kortylewski et al., 2005a).
  • Blocking Stat3 in tumor cells induces tumor cell apoptosis, inhibits tumor angiogenesis and abrogates metastasis (Yu and Jove, 2004; Yu et al., 2007; Xie et al., 2004; Xie et al., 2006), and activates antitumor immune responses (Wang et al., 2004; Kortylewski et al., 2005b).
  • Stat3 is constitutively activated in tumor-stromal myeloid cells, including Grl + immature myeloid cells, DCs, macrophages, NK cell, neutrophils.
  • Activated Stat3 inhibits expression of Th-1 type immune responses while promoting tumor accumulation of T regulatory cells and Thl7 cells, compromising antitumor effects of immune effector cells, such as NK cells, neutrophils and CD8 + T cells (Kortylewski et al., 2005b).
  • Blocking Stat3 in the immune subsets leads to activation of antitumor immunity and immune-mediated tumor growth inhibition and tumor regression (Kortylewski et al., 2005b).
  • Stat3 is a point of convergence for numerous tyrosine kinase signaling pathways, which are the most frequently overactive oncogenic pathways in tumor cells of diverse origins (Yu and Jove, 2004).
  • the reason Stat3 is also constitutively-activated in tumor stromal cells is because many of the Stat3 target genes encode secreted molecules whose cognate receptors signal through Stat3 (Yu et al., 2007).
  • Stat3 -regulated products such as IL-10, IL-6 and VEGF have their receptors in diverse myeloid cells and T lymphocytes.
  • VEGF and bFGF both of which also require Stat3 for their expression, activates Stat3 in endothelial cells.
  • Activated Stat3 promotes expression of a wide range of genes critical for tumor cell survival, proliferation, angiogenesis/metastasis and immune suppression. Activated Stat3 also inhibits expression multiple genes that are pro-apoptotic, anti-angiogenic and Th-1 type immunostimulatory, whose upregulation are critical for anti-cancer therapy (Yu and Jove, 2004; Yu et al., 2007; Kortylewski et al., 2005b).
  • RNA interference provides compelling opportunities to control gene expression in cells and siRNAs therefore represent a family of new drugs with broad potential for the treatment of diverse human diseases.
  • siRNAs therefore represent a family of new drugs with broad potential for the treatment of diverse human diseases.
  • Several recent studies have demonstrated the feasibility of in vivo siRNA delivery, leading to therapeutic effects in mouse models (Song et al., 2005; Hu- Lieskovan et al., 2005; McNamara et al, 2006; Kumar et al., 2007; Poeck et al., 2008) and also in non-human-primates (Li et al., 2005; Zimmermann et al., 2006).
  • aptamers are oligonucleotide-based ligands that bind to specific receptors, such as those on tumor cells. Recent studies further indicated the ability of specific aptamers to bind and modulate the functions of their cognate targets in T cells, leading to potent antitumor immune responses (McNamara et al., 2008). However, whether these aptamers can mediate siRNA delivery into T cells remains to be determined.
  • the immune system can serve as extrinsic tumor suppressor (Bui and Schreiber, 2007; Koebel et al, 2007; Shankaran et al., 2001).
  • the microenvironment of established tumors is typically characterized by a paucity of tumor-specific CD8 + T cells together with an excess of suppressive regulatory T cells and myeloid-derived suppressor cells (MDSC) that promote tumor immune evasion (Kortylewski et al., 2005b; Yu et al, 2005; Curiel et al., 2004; Ghiringhelli et al., 2005; Melani et al., 2003).
  • MDSC myeloid-derived suppressor cells
  • Myeloid cells and other immune cells in the tumor microenvironment also produce growth factors and angiogenic/metastatic factors critical for tumor progression (Kujawski et al., 2008).
  • Stat3 is an important oncogenic molecule. The orchestration of these processes in the tumor microenvironment is highly dependent on the oncogenic transcription factor, Stat3 (Yu et al., 1995; Bromberg et al., 1999; Yu and Jove, 2004; Darnell, 2002; Yu et al., 2007). In particular, we and others have recently demonstrated a critical role of Stat3 in mediating tumor immune evasion (Wang et al., 2004; Kortylewski et al. 2005b; Yu et al., 2007).
  • Activated Stat3 in myeloid cells inhibits expression of a large number of immunostimulatory molecules related to Thl-type responses, while promoting production of several key immunosuppressive factors (Yu et al., 2007, Kortylewski and Yu, 2008; Kortylewski et al., 2009a) as well as angiogenic factors (Kujawski et al., 2008).
  • Stat3 activation in myeloid cells activates Stat3 in tumor cells, enhancing tumor cell proliferation and survival (Bollrath et al., 2009; Grivennikov et al., 2009; Lee et al., 2009; Wang et al., 2009).
  • the present invention relates to methods and compositions for the treatment of diseases, including cancer, infectious diseases and autoimmune diseases.
  • the present invention also relates to methods and compositions for improving immune function.
  • the present invention relates to blocking Stat3, either through genetic knockout, Stat3 small-molecule inhibitor, or Stat3 siRNA, which drastically improves the immune responses induced by CpG.
  • the present invention relates to multifunctional molecules that are capable of being delivered to cells of interest.
  • the multifunctional molecules incorporate an activation element together with a therapeutic element, e.g., a Stat3 blocking element.
  • the multifunctional molecules are capable of being delivered to specific cells of interest including, but not limited to, dendritic cells. These molecules are capable of treating diseases, including cancer, infectious diseases and autoimmune diseases.
  • the present invention is related to chimeric molecules consisting of an active oligonucleotide, such as Toll-like receptor (TLR) ligands, and an active agent, such as double stranded RNA, such as siRNA or activating RNA.
  • TLR Toll-like receptor
  • RNA double stranded RNA
  • the present invention relates to specific chimeric molecules that are useful for the treatment of diseases.
  • the present invention provides a novel molecule for the delivery of an active agent into cells for the treatment of diseases including, but not limited to cancer, infectious diseases and autoimmune diseases.
  • the novel molecules comprises one or more of a first moiety that directs cell or tissue specific delivery of the novel molecule linked to one or more of a second moiety that is an active agent useful for treating cancer or other diseases.
  • the moieties can be linked together directly or they can be linked together indirectly through a linker.
  • the novel molecule comprises two moieties as one molecule that is multifunctional. For example, a TLR ligand and an siRNA are made into one molecule for delivery, immune stimulation and blocking immunosuppressive elements, such as Stat3, and/or oncogenic effects, such as caused by Stat3.
  • the novel molecule comprises multifunctional moieties attached to a linker, such that it can contain a multitude of moieties.
  • the linker is bifunctional producing a molecule of the structure A-X-B, where X is a linker, one of A and B is a moiety that is capable of delivering the molecule to cells of interest and the other one of A and B is an active agent useful for treating the cancer or other disease.
  • a and/or B may also be subject to further linking.
  • the linker is multifunctional, producing a molecule having more than two moieties. In one embodiment, using as an example a quadrifunctional form, such a molecule can have the structure
  • the active agent is a double stranded RNA molecule that either downregulates gene expression, such as an siRNA molecule, or activates gene expression, such as an activating RNA molecule.
  • the active agent is a small molecule drug or peptide.
  • the delivery moiety is a ligand for a toll-like receptor (such as oligonucleotides described herein).
  • the delivery moiety is another cell-specific ligand (including, but not limited to, aptamers).
  • the binding sites on a linker may be specific for each type of moiety to be linked, for example a linker with a structure that has one region capable of likening to an oligonucleotide and another region capable of binding to a peptide.
  • Other variations of structure can be proposed by utilizing structures and linkers that promote branching, circularization or linearization of the molecules, including combinations thereof.
  • Any element of a multimeric molecule, including the linker may also have additional functional properties such as being a substrate for chemical reactions, including enzyme catalyzed reactions, lability in environmental conditions such as oxygen tension, pH, ionic conditions.
  • any element of a multimeric molecule may also include labels to promote detection - using active or passive detection of electromagnetic emissions (e.g. optical, ultraviolet, infra-red), radioactivity, magnetic resonance or ability to be cleaved or catalyse a reaction.
  • electromagnetic emissions e.g. optical, ultraviolet, infra-red
  • radioactivity e.g. radioactivity
  • magnetic resonance e.g. magnetic resonance
  • many means are available to promote this including use of fluorochromes, quantum dots, dyes, inherent physical chemical properties structures such as spectral absorbance or emission characteristics magnetic resonance enhancers, and radioisotopes.
  • the present invention provides a method for the treatment of diseases (including, but not limited to, cancer, infectious diseases, autoimmune diseases, diseases due to excessive angiogenesis and diseases that can benefit from increased angiogenesis) which comprises using the novel molecules of the present invention.
  • diseases including, but not limited to, cancer, infectious diseases, autoimmune diseases, diseases due to excessive angiogenesis and diseases that can benefit from increased angiogenesis.
  • the molecules of the present invention are administered to patients in need of treatment using conventional pharmaceutical practices.
  • the present invention provides active agents that are capable of acting in the Stat3 pathway which, when taken up by the cells of interest, results in the treatment of diseases including, but not limited to, cancer, infectious diseases and autoimmune diseases.
  • FIGs 1A-1D show that CpG-STAT3 siR A approach effectively silences genes in TLR9+ human acute myeloid leukemia (AML) cells, leading to therapeutic antitumor effects in xenotransplanted tumor models in mice.
  • Fig. 1A NOD/SCID/IL-2Rynull (NSG) mice were injected i.v. with 107 of human MV4-11 leukemia cells. Four weeks later, mice with engrafted AML cells were injected i.v. with the 100 ⁇ g dose of various CpG(A)-siRNAs daily for three days.
  • Fig. IB CpG(A)-STAT3 siRNA in vivo treatment leads to STAT3 gene silencing (left, by qPCR), tumor cell death (middle, by FACS analysis of Annexin V- positive tumor cell suspensions) and reduced growth rate of human myeloma tumors in NSG mice (right).
  • Tumors were treated with daily intratumoral injections of 20 ⁇ g CpG-STAT3 siRNA starting 4-5 days after injection of 107 of tumor cells (at the average tumor size 10 mm).
  • Blocking oiSTAT3 in MonoMac6 cells (Fig. 1C) and BCL-XL in MV4-11 AML cells (Fig. ID) in vivo inhibits growth of xenotransplanted tumors in NSG mice.
  • the target gene silencing left graphs in Fig. 1C, ID
  • tumor cell death middle graph in Fig. ID
  • tumor growth kinetics right panels
  • FIG. 2 shows the efficacy of in vivo target gene silencing by CpG-STAT3 siRNA depends on the CpG ODN sequence.
  • Fig. 2 top NOD/SCID/IL-2Rynull (NSG) mice were injected s.c. with 5x106 of human MV4-11 leukemia cells. Tumors were treated with two daily intratumoral injections of 20 g various CpG-siRNAs as indicated, including CpG-Luciferase siRNA and CpG-STAT3 siRNA in two versions, conjugated to class A (D19 ODN) or class B (7909) CpG ODN.
  • the STAT3 gene silencing was assessed by quantitative real-time PCR (Fig. 2 top), while tumor cell death was measured by FACS analysis using Annexin V staining of tumor cell suspensions (Fig. 2 bottom). Shown are the representative results from a single experiments using 5-6 mice per each experimental group; means ⁇ s.e.m.
  • Figure 3 shows that the class A ODN-based CpG(O19)-STAT3 siRNA conjugates induce production of proinflammatory protein mediators without stimulating expression of potentially tumor promoting IL-6, IL-8 or IL-10, which are co-activated by two other CpG- siRNA types.
  • Human PBMCs were incubated for 24 h in the presence of class A - CpG(D19)- STAT3 siRNA, calls B - CpG(7909)-STAT3 siRNA or class C - CpG(2429)-STAT3 siRNA conjugates in concentrations as indicated.
  • FIG. 4 shows that the CpG(Ol9)-STAT3 siRNA does not induce exacerbated type I interferon response, in contrast to unconjugated D19 class A oligodeoxynocleotides.
  • Human PBMCs were incubated for 24 h in the presence of STAT3 siRNA, CpG(A)-D19, CpG(B)-7909 alone or as CpG-STAT3 siRNA conjugates in concentrations as indicated.
  • Supernatants from cultured PBMCs were analyzed for the IFNa production using Cytokine Bead Array on Luminex platform. Shown are representative results from one of two independent experiment performed in triplicates; ND - not detectable.
  • the present invention relates to methods and compositions for the treatment of diseases. More particularly, the present invention relates to multifunctional molecules that are capable of being delivered to cells of interest for the treatment of diseases including, but not limited to, cancer, infectious diseases and autoimmune diseases. More specifically, the present invention relates to specific chimeric molecules that are useful for the treatment of diseases.
  • the present invention provides a novel molecule for the delivery of an active agent into cells for the treatment of cancer and other diseases including, but not limited to infectious diseases and autoimmune diseases.
  • the novel molecules comprises one or more of a first moiety that directs cell or tissue specific delivery of the novel molecule linked to one or more of a second moiety that is an active agent useful for treating cancer or other diseases.
  • the moieties can be linked together directly or they can be linked together indirectly through a linker.
  • the novel molecule comprises two moieties as one molecule that is multifunctional. For example, a TLR ligand and an siRNA are made into one molecule for delivery, immune stimulation and blocking immunosuppressive elements, such as Stat3, and/or oncogenic effects, such as caused by Stat3.
  • the novel molecule comprises moieties attached to a linker that is multifunctional, such that it can contain a multitude of moieties.
  • the linker is bifunctional producing a molecule of the structure A-X-B, where X is a linker, one of A and B is a moiety that is capable of delivering the molecule to cells of interest and the other one of A and B is an active agent useful for treating the cancer or other disease.
  • the linker is a modification of, or structure present on, either moiety A or B, or both, that results in a binding between the two elements. The binding maybe covalent or non-covalent bonds.
  • the linker is multifunctional, for example, quadrifunctional, producing a molecule having more than two moieties. In one embodiment, such a molecule can have the structure
  • X is the linker
  • one or more of A, B, Y and Z is a moiety that is capable of delivering the molecule to cells of interest and the others are an active agent useful for treating the cancer or other disease.
  • the linker may have any number of other moieties attached to it, and the examples of having two or four moieties, and their lack of any secondary extension, for example a modification of Y, is merely for illustration purposes and not intended to be limiting.
  • the active agent is a double stranded RNA molecule that either downregulates gene expression, such as a siRNA molecule, or activates gene expression, such as an activating RNA molecule.
  • the active agent is a small molecule drug or peptide.
  • the delivery moiety is a ligand for a toll-like receptor (such as oligonucleotides described herein).
  • the delivery moiety is another cell- specific ligand (such as aptamers).
  • the present invention provides a method for the treatment of diseases which comprises using the novel molecules of the present invention.
  • Diseases which can be treated in accordance with the present invention include cancer, infectious diseases, autoimmune diseases, diseases due to excessive angiogenesis and diseases that can benefit from increased angiogenesis.
  • Cancers which can be treated with the molecules of the present invention include, but are not limited to, melanoma, skin cancer, precancerous skin lesions, breast cancer, prostate cancer, lung cancer, glioma, pancreatic cancer, head and neck cancer, multiple myeloma, leukemias, lymphomas.
  • infectious diseases include, but are not limited to, HIV, HPV infection and hepatitis.
  • autoimmune diseases include, but are not limited to, psoriasis, multiple sclerosis (MS) and inflammatory bowel disease (IBD).
  • diseases due to excessive angiogenesis include, but are not limited to, cancer, diabetic retinopathy and Kaposi's Sarcoma.
  • diseases that can benefit from increased angiogenesis include, but are not limited to, diseases needing wound repair (healing).
  • the molecules of the present invention are administered to patients in need of treatment using conventional pharmaceutical practices.
  • the present invention provides active agents that are capable of acting in the Stat3 pathway which, when taken up by the cells of interest, results in the treatment diseases including, but not limited to cancer, infectious diseases and autoimmune diseases.
  • the molecules of the present invention have several advantages that result from the characteristics of the molecules. These advantages include:
  • oligonucleotide or “oligo” shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)).
  • oligonucleotide refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs).
  • oligonucleotide shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
  • a "stabilized oligonucleotide” shall mean an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease).
  • Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone.
  • Especially preferred oligonucleotides have a phosphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur).
  • oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • CpG containing oligonucleotide refers to an oligonucleotide, which contains a cytosine/guanine dinucleotide sequence.
  • Preferred CpG oligonucleotides are between 2 to 100 base pairs in size and contain a consensus mitogenic CpG motif represented by the formula:
  • C and G are unmethylated, X 1; X 2 , X 3 and are nucleotides and a GCG trinucleotide sequence is not present at or near the 5' and 3' ends.
  • Examples of CpG ODNs are described in U.S. Patent Nos. 6,194,388 and 6,207,646, each incorporated herein by reference.
  • the CpG oligonucleotides range between 8 and 40 base pairs in size.
  • the CpG oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides.
  • the CpG ODNs or CpG ORNs can be synthesized as an oligonucleotide. Alternatively, CpG ODNs or CpG ORNs can be produced on a large scale in plasmids.
  • An "aptamer” refers to a nucleic acid molecule that is capable of binding to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, 1990; Ellington and Szostak, 1990).
  • the binding of a ligand to an aptamer which is typically RNA, changes the conformation of the aptamer and the nucleic acid within which the aptamer is located.
  • the conformation change inhibits translation of an mRNA in which the aptamer is located, for example, or otherwise interferes with the normal activity of the nucleic acid.
  • Aptamers may also be composed of DNA or may comprise non-natural nucleotides and nucleotide analogs.
  • An aptamer will most typically have been obtained by in vitro selection for binding of a target molecule. However, in vivo selection of an aptamer is also possible. An aptamer will typically be between about 10 and about 300 nucleotides in length. More commonly, an aptamer will be between about 30 and about 100 nucleotides in length. See, e.g., U.S. Patent No. 6,949,379, incorporated herein by reference. Examples of aptamers that are useful for the present invention include, but are not limited to, PSMA aptamer (McNamara et al.,
  • CTLA4 aptamer Surveli-Marotto et al., 2003
  • 4- IBB aptamer Surveli-Marotto et al.
  • TLR Toll-like receptor
  • TLR1- TLR10 highly conserved mammalian pattern recognition receptor proteins
  • PAMPs pathogen-associated molecular patterns
  • TLR polypeptides share a characteristic structure that includes an extracellular (extracytoplasmic) domain that has leucine-rich repeats, a transmembrane domain, and an intracellular (cytoplasmic) domain that is involved in TLR signaling.
  • TLRs include, but are not limited, to human TLRs.
  • TLRs include, but are not limited to TLR9, TLR8 and TLR3.
  • TLR ligand or "ligand for a TLR” refer to a molecule, that interacts, directly or indirectly, with a TLR through a TLR domain and is capable of being internalized by cells.
  • a TLR ligand is a natural ligand, i.e., a TLR ligand that is found in nature.
  • a TLR ligand refers to a molecule other than a natural ligand of a TLR, e.g., a molecule prepared by human activity, such as a CpG containing oligonucleotide.
  • target cells for ODN- or ORN-mediated delivery include any cell that is capable of internalizing a TLR ligand.
  • Such cells include (a) cells of the myeloid lineage including dendritic cells, macrophages and monocytes, (b) cells of the lymphoid lineage including B cells and T cells, (c) endothelial cells and (d) malignant cells being derivatives of the previously mentioned cells, e.g., multiple myeloma, B cell lymphoma and T cell lymphoma.
  • the malignant cells can also be any cells that possess the capacity of uptaking and/or internalizing a TLR ligand.
  • novel molecules are provided by an active moiety for delivering an active agent to a cell of interest for the treatment of diseases as disclosed herein.
  • the novel molecules comprises one or more of a first moiety that directs cell or tissue specific delivery of the novel molecule linked to one or more of a second moiety that is an active agent useful for treating cancer or other diseases.
  • the moieties can be linked together directly or they can be linked together indirectly through a linker.
  • the novel molecule comprises two moieties as one molecule that is multifunctional.
  • a TLR ligand and an siRNA are made into one molecule for delivery, immune stimulation and blocking immunosuppressive elements, such as Stat3, and/or oncogenic effects, such as caused by Stat3.
  • the novel molecule comprises moieties attached to a linker that is multifunctional, such that it can contain a multitude of moieties.
  • the linkage of the first and second moieties can be provided through diverse structures and/or chemistry.
  • the linkage can also be designed to allow for one first moiety to be linked to multiple second moieties.
  • the linkage can be designed to allow for linkage of a first moiety to small molecule drugs or peptides.
  • the molecule may have the structure A-X-B. In another embodiment, the molecule may have the structure
  • X is a linker between the A and B moieties or between the A, B, Y and Z moieties.
  • X may be multifunctional reactive molecule having, e.g., NP, where N is a nucleic acid binding sites and P is a peptide binding site.
  • the linker may be derivatized, e.g., with FITC, such that the X moiety itself is also functional.
  • X may be derivatized with a fluorochrome or similar molecule, or may be derivatized with a chemotherapeutic agent.
  • A, B, etc. i.e., any moiety attached to the linker
  • the function of A, B, etc. can be selected to include from delivery (including approaches to target to cells, tissues, organs), improved pharmacokinetic properties, cytotoxic, cytostatic, apoptotic, gene modulating (including upregulation, e.g., activating RNA, or downregulation, e.g., siRNA), proinflammatory, anti-inflammatory, antigenic, immunogenic pro-coagulant, anti-coagulant properties, pro-drug elements and combinations thereof.
  • each of these moieties can modified as known in current state of art to improve their desired properties.
  • These (A, B or desired modifications) can also be selected for via screening, evolution or combinatorial approaches as is well known to the skilled artisan.
  • moieties that can be used for delivery include CpG ODNs, CpG ORNs, polyG (Peng et al., 2005), poly(I:C) (Alexopoulou et al, 2001) (such as ligands for tolllike receptors (TLRs)) and aptamers.
  • the TLR ligands are useful for delivering the molecules of the present invention to cells that are capable of internalizing TLR ligands.
  • Aptamers are useful for delivering the molecules of the present invention to cells which specifically bind the aptamers.
  • some elements or moieties may be themselves bifunctional or derivatized to be bifunctional or have improved function (e.g., adding a 5' triphosphate on a CpG may be an enhanced stimulator of intracellular and/or extracellular signaling).
  • a molecule of the present invention is prepared by linking a first moiety, e.g. a CpG ODN, CpG ORN, oligonucleotides or aptamer, to a second moiety, e.g., a dsRNA, using multiple units of the C3 spacer as the linker (Dela et al., 1987).
  • a method for preparing such a molecule in which the first moiety is a CpG ODN is shown in the Examples.
  • a molecule of the present invention can be prepared by providing a dsRNA in which one of the strands has an overhang and the first moiety has a complementary overhang.
  • the overhang can be spaced from the first moiety and the dsRNA by using linkers comprising multiple units of the C3 spacer. After annealing, both components are connected creating a desired construct. By controlling the length of the overhang and its makeup we can control the strength and the specificity of the attachment.
  • the preferred component of the overhang are: 2'-0-methyl RNA (2'-OMe), 2'-Fluoro RNA (2'-F) or Locked Nucleic Acids (LNAs) or PNA. Extremely high melting temperatures of an LNA/LNA duplex allow for the use of much shorter overhangs. 2'-Fluoro RNA (2'-F) were reported to have lower toxicity then 2'-0-methyl RNA (2'-OMe). Since the cost of LNA is still 10-15 times higher then 2'-Fluoro RNA (2'-F) the latter seems to be the optimal choice for overhang component. Use of all of the above increases the resistance of the oligonucleotide to cellular nucleases.
  • the other exemplary sugar modifications include, for example, a 2'-0-methoxyethyl nucleotide, a 2 -0- NMA, a 2'-DMAEOE, a 2'-AP, 2'-hydroxy, or a 2'-ara-fluoro or extended nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA).
  • ENA extended nucleic acid
  • HNA hexose nucleic acid
  • CeNA cyclohexene nucleic acid
  • overhangs for the construction allows for: (i) use of smaller molecules, (ii) higher purity at lower cost, (iii) lower cost of final product and (iv) flexibility (construction of product on demand; possibility of matching of one component with multiple components).
  • the use of a universal overhangs allows for the interchangeability of the components.
  • branching or bridging compounds allows for the synthesis of a component carrying two or more overhangs.
  • Such branching or bridging compounds allows for the attachment of multiple first moiety components, e.g., CpG ODN, to the second moiety component, e.g., dsR A, and/or for the attachment of multiple second moiety components to the multiple first moiety components.
  • the use of molecules having in multiple overhangs allows for the assembly of complementary constructs consisting of two or more aptamers. Constructs of this kind would be used in the dimerization experiments.
  • the use of molecules having multiple overhangs allows for the assembly of complementary constructs consisting of an aptamer and two or more siRNA duplexes.
  • Covalent constructs can also be prepared to form the molecules of the present invention.
  • the first and second moieties have reactive groups.
  • a covalent bond is created during the chemical reaction between the reactive groups. Examples of such pairs of the reactive groups are as follows.
  • (B) azide and acetylene groups combine readily with each other—when held in close proximity—to form triazoles.
  • Click chemistry is the use of chemical building blocks with "built-in high-energy content to drive a spontaneous and irreversible linkage reaction with appropriate complementary sites in other blocks," Use of the azide-acetylene reaction represents “true progress” because of its high selectivity.
  • Vinyl sulfones and substituted divinyl sulfones readily react with sulfuhydryl group (SH) in pH 5-7, with and terminal phosphothioesters in pH7, and with primary and secondary amines at higher pH.
  • Conjugation of two biopolymers with the use of click chemistry can also be used to create the molecules of the present invention.
  • Reaction of dsRNA component having multiple reactive groups with the excess of the CPG or aptamer component leads to the products consisting of multiple dsRNAs attached to the single CPG or aptamer component.
  • Reaction of first moiety having multiple reactive groups with the excess of the small molecule drug leads to the products consisting of multiple drug molecules attached to a single CpG or aptamer component.
  • Drugs may be attached to the constructs through the hydrolysable-digestible linker, such as a short peptide hydrolysable by esterase, to facilitate its release upon delivery to the target.
  • the active agents of the present invention are double stranded RNA molecules. These double stranded RNA molecules may be useful for downregulating gene expression, such as siRNA molecules. Alternatively, the double stranded RNA molecules may be useful for upregulating gene transcription, such as activating RNA molecules.
  • the siRNA molecule may have different forms, including a single strand, a paired double strand (dsRNA) or a hairpin (shRNA) and can be produced, for example, either synthetically or by expression in cells.
  • DNA sequences for encoding the sense and antisense strands of the siRNA molecule to be expressed directly in mammalian cells can be produced by methods known in the art, including but not limited to, methods described in U.S. published application Nos. 2004/0171118 Al, 2005/0244858 Al and 2005/0277610 Al, each incorporated herein by reference.
  • the siRNA molecules are coupled to carrier molecules, such as CpG oligonucleotides, various TLR-ligands (such as polyG or poly(I:C) or RNA aptamers, using the techniques known in the art or described herein.
  • DNA sequences encoding a sense strand and an antisense strand of a siRNA specific for a target sequence of a gene are introduced into mammalian cells for expression.
  • mammalian cells may be exposed to multiple siRNAs that target multiple sequences in the gene.
  • the siRNA molecules generally contain about 19 to about 30 base pairs, and may be designed to cause methylation of the targeted gene sequence. In one embodiment, the siRNA molecules contain about 19-23 base pairs, and preferably about 21 base pairs. In another embodiment, the siRNA molecules contain about 24-28 base pairs, and preferably about 26 base pairs. In a further embodiment, the dsRNA has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2 base 3 '-overhang. See, for example, U.S. published application Nos. 2005/0244858 Al, 2005/0277610 Al and 2007/0265220 Al, each incorporated herein by reference.
  • this dsRNA having an asymmetric structure further contains 2 deoxynucleotides at the 3 'end of the sense strand in place of two of the ribonucleotides.
  • Individual siRNA molecules also may be in the form of single strands, as well as paired double strands ("sense” and "antisense") and may include secondary structure such as a hairpin loop. Individual siRNA molecules could also be delivered as precursor molecules, which are subsequently altered to give rise to active molecules. Examples of siRNA molecules in the form of single strands include a single stranded anti-sense siRNA against a non-transcribed region of a DNA sequence (e.g. a promoter region).
  • the sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the dsRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3 'end of antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
  • the RNAi molecule may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21mer and (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings.
  • a "typical” 21mer siRNA is designed using conventional techniques, such as described above. This 21mer is then used to design a right shift to include 1-7 additional nucleotides on the 5' end of the 21mer. The sequence of these additional nucleotides may have any sequence.
  • the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the siRNA is not required. That is, the resultant siRNA is sufficiently complementary with the target sequence.
  • the first and second oligonucleotides are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence.
  • the dsRNA has an asymmetric structure, with the antisense strand having a 25-base pair length, and the sense strand having a 27-base pair length with a 2 base 3 '-overhang.
  • this dsRNA having an asymmetric structure further contains 2 deoxynucleotides at the 3 'end of the antisense strand.
  • Suitable dsRNA compositions that contain two separate oligonucleotides can be linked by a third structure.
  • the third structure will not block Dicer activity on the dsRNA and will not interfere with the directed destruction of the R A transcribed from the target gene.
  • the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used.
  • the third structure may be an oligonucleotide that links the two oligonucleotides of the dsRNA is a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsRNA composition. The hairpin structure will not block Dicer activity on the dsRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene.
  • the sense and antisense sequences may be attached by a loop sequence.
  • the loop sequence may comprise any sequence or length that allows expression of a functional siRNA expression cassette in accordance with the invention.
  • the loop sequence contains higher amounts of uridines and guanines than other nucleotide bases.
  • the preferred length of the loop sequence is about 4 to about 9 nucleotide bases, and most preferably about 8 or 9 nucleotide bases.
  • the dsRNA i.e., the RNAi molecule
  • the dsRNA has several properties which enhances its processing by Dicer.
  • the dsRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the dsRNA has a modified 3 ' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the longest strand in the dsRNA comprises 24-30 nucleotides.
  • the sense strand comprises 24-30 nucleotides and the antisense strand comprises 22-28 nucleotides.
  • the resulting dsRNA has an overhang on the 3 ' end of the sense strand.
  • the overhang is 1-3 nucleotides, such as 2 nucleotides.
  • the antisense strand may also have a 5' phosphate.
  • Modifications can be included in the dsRNA, i.e., the RNAi molecule, so long as the modification does not prevent the dsRNA composition from serving as a substrate for Dicer.
  • one or more modifications are made that enhance Dicer processing of the dsRNA.
  • one or more modifications are made that result in more effective RNAi generation.
  • one or more modifications are made that support a greater RNAi effect.
  • one or more modifications are made that result in greater potency per each dsRNA molecule to be delivered to the cell.
  • Modifications can be incorporated in the 3 '-terminal region, the 5 '-terminal region, in both the 3 '-terminal and 5'-terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind any number and combination of modifications can be incorporated into the dsRNA. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5 '-terminus can be phosphorylated.
  • the antisense strand is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the dsRNA is designed to direct orientation of Dicer binding and processing.
  • suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like.
  • Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs.
  • nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2*,3'- didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2 ⁇ 3'- dideoxythymidine (d4T).
  • deoxynucleotides are used as the modifiers.
  • nucleotide modifiers When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand.
  • sterically hindered molecules When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers.
  • the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing.
  • two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3 '-end of the sense strand.
  • This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.
  • modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like.
  • modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-0-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003).
  • base groups examples include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Patent Nos. 5,684,143, 5,858,988, 6,291,438 and 7,307,069 and in U.S. published patent application No. 2004/0203145 Al, each incorporated herein by reference. Other modifications are disclosed in Herdewijn (2000), Eckstein (2000), Rusckowski et al. (2000), Stein et al. (2001) and Vorobjev et al. (2001), each incorporated herein by reference.
  • the siRNA structure can be optimized to ensure that the oligonucleotide segment generated from Dicer's cleavage will be the portion of the oligonucleotide that is most effective in inhibiting gene expression.
  • a 27- bp oligonucleotide of the dsRNA structure is synthesized wherein the anticipated 21 to 22-bp segment that will inhibit gene expression is located on the 3 '-end of the antisense strand.
  • the remaining bases located on the 5 '-end of the antisense strand will be cleaved by Dicer and will be discarded.
  • This cleaved portion can be homologous (i.e., based on the sequence of the target sequence) or non-homologous and added to extend the nucleic acid strand.
  • Activating RNA molecules are similar in design as siRNA molecules. However, they can also be shorter than siRNA molecules. Thus, activating RNA molecules may be 12-30 nucleotides in length, although a length of 18-30 nucleotides is preferred. Activating RNA molecules are targeted to the promoter region of the gene of interest and are designed to induce transcriptional activation. In one embodiment, the region within the promoter of the gene is selected from a partially single-stranded structure, a non-B-DNA structure, an AT-rich sequence, a cruciform loop, a G-quadruplex, a nuclease hypersensitive elements (NHE), and a region located between nucleotides -100 to +25 relative to a transcription start site of the gene.
  • NHE nuclease hypersensitive elements
  • RNA for the siR A or activating R A component of the present invention may be produced enzymatically or by partial/total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis.
  • each strand is prepared chemically.
  • the active agents of the present invention are small molecule drugs or peptides.
  • small molecule drugs include, but are not limited to, Stat3 inhibitors (such as those commercially available from Calbiochem), Imatinib (Bcr-Abl), Sunitib (VEGF receptor), Sorefenib (Raf) and DASATINIB (Src).
  • peptides include, but are not limited to, Stat3 peptidomimetics, p53 peptidomimetics and Farnesyl Transferase inhibitors.
  • the present invention further provides active agents that are capable of acting in the Stat3 signaling pathway or affecting genes regulated by Stat3. These active agents, when taken up by the cells of interest, result in the treatment of cancer or other diseases.
  • the active agent is an siRNA molecule directed against Stat3 and results in the down regulation of Stat3.
  • the active agent is an siRNA molecule directed against SOCS3 which is an inhibitor of Stat3.
  • the active agent is an activating RNA for tumor suppressor genes.
  • the present invention provides a method for treating diseases.
  • the molecules of the present invention are administered to patients in need of treatment using conventional pharmaceutical practices. Suitable pharmaceutical practices are described in Remington: The Science and Practice of Pharmacy, 21 st Ed., University of Sciences in Philadelphia, Ed., Philadelphia, 2005.
  • the present invention provides for the delivery of dsRNA, such as siRNA or activating RNA, for the treatment of cancer.
  • the present invention provides for the delivery of dsRNA for the treatment of infectious diseases.
  • the present invention provides the delivery of dsRNA for the treatment of autoimmune diseases.
  • the dsRNA can be specifically delivered to cells as described herein.
  • the present invention can also be used to deliver DNA or RNA that encode antigens to cells, e.g., DCs to stimulate an immune response, e.g., vaccme or immunomodulator.
  • Suitable antigens could be tumor or infectious agents, including but not limited to, virus, fungus, bacteria, rikettsia, amoeba.
  • the present invention relates to the use of multifunctional molecules to modulate cancer and the immune system.
  • the present invention relates delivery of RNA (siR A and/or activating R A) by TLR ligands as single molecule in vivo.
  • the present invention is illustrated herein by a covalently linked siRNA and CpG molecule.
  • siRNA siRNA
  • CpG covalently linked siRNA
  • Other TLR ligands including but not limited to polyI:C, polyG LPS, and peptidoglycan can also been linked to siRNAs for various target genes.
  • Stat3 is a 'master switch'- in both cancer and tumor cells and tumor-associated immune cells - that controls tumor survival, angiogenesis/metastasis and immune evasion.
  • the challenge is to turn Stat3 off in the desired cells in cancer in patients.
  • the present invention describes the development of optimal Stat3 siRNAs (Dicer) with antitumor effects in vivo, and shows that Stat3 siRNA linked to CpG oligonucleotide efficiently enters dendritic cells. Targeting Stat3 drastically improves CpG-based cancer.
  • the utility of the present invention has been demonstrated herein using melanoma as the model. However, it is understood that the present invention is not limited to melanoma but is equally applicable to all types of cancer.
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising of molecules of the present invention, i.e., the molecules that contain a cell specific delivery moiety and one or more additional active agents.
  • the cell specific delivery moiety and the additional active agent(s) may be directly linked together or they may be indirectly linked together through the use of a linker.
  • the active agent may be an siRNA, an activating RNA, a small molecule drug or a peptide.
  • siRNA can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
  • buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
  • cationic lipids such as lipofectin (U.S. Patent No.
  • Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, CO), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.
  • the method of introducing the molecules of the present invention into the environment of the cell will depend on the type of cell and the make up of its environment.
  • a lipid formulation such as in lipofectamine and the molecules of the present invention can be added directly to the liquid environment of the cells.
  • Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
  • the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable.
  • Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.
  • molecules of the present invention in a buffer or saline solution and directly inject the formulated dsRNA into cells, as in studies with oocytes.
  • the direct injection of dsRNA duplexes may also be done.
  • suitable amounts of molecules of the present invention must be introduced and these amounts can be empirically determined using standard methods.
  • effective concentrations of individual dsRNA species in the environment of a cell will be about 50 nanomolar or less 10 nanomolar or less, or compositions in which concentrations of about 1 nanomolar or less can be used.
  • methods utilize a concentration of about 200 picomolar or less and even a concentration of about 50 picomolar or less can be used in many circumstances.
  • effective doses of small molecule drugs or peptides can be lower than previously used in view of the cell specific delivery provided by the present invention.
  • the method can be carried out by addition of the compositions containing the molecules of the present invention to any extracellular matrix in which cells can live provided that the composition is formulated so that a sufficient amount of the active agent can enter the cell to exert its effect.
  • the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.
  • Expression of a target gene can be determined by any suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure the expression of a target gene will depend upon the nature of the target gene. For example, when the target gene encodes a protein the term "expression" can refer to a protein or transcript derived from the gene. In such instances the expression of a target gene can be determined by measuring the amount of mRNA corresponding to the target gene or by measuring the amount of that protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where the gene product is an RNA species expression can be measured by determining the amount of RNA corresponding to the gene product. The measurements can be made on cells, cell extracts, tissues, tissue extracts or any other suitable source material.
  • the determination of whether the expression of a target gene has been reduced can be by any suitable method that can reliably detect changes in gene expression. Typically, the determination is made by introducing into the environment of a cell undigested siRNA such that at least a portion of that siRNA enters the cytoplasm and then measuring the expression of the target gene. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared. Similarly the determination can be made by introducing into the environment of a cell undigested activating RNA such that at least a portion of that activating RNA enters the cytoplasm and then measuring the expression of the target gene.
  • the molecules of the present invention can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of the molecules and pharmaceutically acceptable carrier.
  • a pharmacologically or therapeutically effective amount refers to that amount of a molecule of the present invention effective to produce the intended pharmacological, therapeutic or preventive result.
  • the phrases "pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to that amount of a dsRNA, small molecule drug or peptide effective to produce the intended pharmacological, therapeutic or preventive result.
  • a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • the pharmaceutically acceptable carrier of the disclosed dsRNA composition may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.
  • Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991).
  • the polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution
  • compositions of this invention can be administered by any means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection
  • a suitable dosage unit of active agent moiety of the molecules of the present invention will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day.
  • Pharmaceutical composition comprising the siRNA can be administered once daily.
  • the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • the active agent, e.g., dsRNA, contained in each sub- dose must be correspondingly smaller in order to achieve the total daily dosage unit.
  • the dosage unit can also be compounded for a single dose over several days, e.g. , using a conventional sustained release formulation which provides sustained and consistent release of the active agent, e.g., dsRNA, over a several day period. Sustained release formulations are well known in the art.
  • the dosage unit contains a corresponding multiple of the daily dose.
  • the pharmaceutical composition must contain active agent, e.g., dsRNA, in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated.
  • the composition can be compounded in such a way that the sum of the multiple units of active agent together contain a sufficient dose.
  • Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans.
  • the dosage of compositions of the invention lies within a range of circulating concentrations that include the ED 50 (as determined by known methods) with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels of dsRNA in plasma may be measured by standard methods, for example, by high performance liquid chromatography.
  • the present invention relates to a method for TGS in a mammalian, including human, cell.
  • the method comprises introducing the siRNA containing molecules of the present invention into the appropriate cell.
  • introducing encompasses a variety of methods of introducing the siRNA containing molecules into a cell, either in vitro or in vivo, such as described above.
  • the present invention relates to a method for gene activation in a mammalian cell, including human cell.
  • the method comprises introducing the activating RNA containing molecules of the present invention into the appropriate cell.
  • introducing encompasses a variety of methods of introducing the siRNA containing molecules into a cell, either in vitro or in vivo, such as described above.
  • the present invention relates to a method for treating a disease or physiological disorder or condition in a mammal, including a human.
  • the method comprises introducing the small molecule drug or peptide containing molecules of the present invention into the appropriate cell.
  • introducing encompasses a variety of methods of introducing the siRNA containing molecules into a cell, either in vitro or in vivo, such as described above.
  • TLR ligands such as CpG
  • CpG CpG
  • blocking Stat3 either genetically, or pharmacologically, results in drastically improved immune responses and antitumor effects.
  • TLR ligand e.g., a moiety consisting of oligonucleotides that can activate immune responses against cancer and infectious diseases when it is linked to siRNA
  • a TLR ligand e.g., a moiety consisting of oligonucleotides that can activate immune responses against cancer and infectious diseases when it is linked to siRNA
  • a TLR ligand e.g., a moiety consisting of oligonucleotides that can activate immune responses against cancer and infectious diseases when it is linked to siRNA
  • myeloid cells such as macrophages and dendritic cells
  • This uptake occurs in the absence of any transfection agents.
  • the DNA- RNA chimeric constructs can be processed by Dicer and is associated with Dicer in living cells.
  • CpG-siRNA chimeric constructs can be efficiently taken up by both human and mouse B cell malignant cells (B cell lymphoma and multiple myeloma).
  • Stat3 is a potent oncogenic transcriptional factor that is continuously activated in diverse human cancer (Yu and Jove, 2004). Activated Stat3 not only promotes tumor cell survival, proliferation and angiogenesis (Yu and Jove, 2004), it also mediates tumor immune suppression through its activation in both tumor cells and in immune cells in the tumor microenvironment (Wang et al., 2004; Kortylewski et al., 2005b; Yu et al., 2007). Effective targeting of Stat3 in tumor cells has been shown to induce tumor cell apoptosis, inhibit tumor cell proliferation, angiogenesis/metastasis (Yu and Jove, 2004).
  • CpG is a potent immune stimulator, its effects in tumor-bearing hosts are dampened by the tumor microenvironment, which is, at least in part, mediated by Stat3 activation.
  • CpG like several other pathogen-associated immune stimulators, such as LPS, is an activator of Stat3 (through activating IL-10, which in turn activates Stat3), and Stat3 serves as feedback mechanism to limit their immunostimulatory effects (Benkhart et al., 2000; Samarasinghe et al., 2006).
  • siRNA against Stat3 (SEQ ID NO:3 for sense strand; SEQ ID NO:2 for antisense strand) is linked to toll-like receptor 9 ligand, CpG oligonucleotide (ODN) (SEQ ID NO:l).
  • ODN CpG oligonucleotide
  • Optimal sequences of both human and mouse Stat3 siRNA have been selected, followed by linkage to CpG single stranded ODN, and other toll-like receptor ligands.
  • the construct can be processed by Dicer, and is associated with Dicer in living cells, and causes gene silencing.
  • the chimeric constructs when delivered in vivo in tumor bearing mice, are efficiently uptaken and internalized by targeted cells, such as macrophages and dendritic cells. These immune cells are able to traffic from tumor to tumor training lymph nodes, where they can interact with T cells. In vivo gene silencing is also detected in dendritic cells and macrophages in tumor draining lymph nodes.
  • the immune modulation induced by the toll-like receptor 9 Iigand-Stat3 siRNA leads to potent antitumor effects on well established B16 melanoma. Both local intratumoral injection and systemic intravenous injection routes are tested, demonstrating the usefulness of the ODN-siRNAs as therapeutic agents.
  • CpG alone, Stat3siR A alone, or CpG-linked to a scrambled siRNA are not able to induce significant antitumor effects, testifying the superior efficacies of linking two active moieties: TLR9 ligand and Stat3 siRNA.
  • Tumor bearing mice treated with the CpG- Stat3siRNA constructs display activation of dendritic cells, increased CD8+ T cells, NK cells and reduced number of T regulatory cells in the tumor and/or tumor draining lymph nodes. Treating tumor-bearing mice with CpG-Stat3 siRNA also increases tumor infiltrating tumor antigen-specific CD8+ T cells. See U.S. Patent Application Publication No. 2008/021443 6, PCT International Publication No. WO 2008/094254, and U.S. Patent Application Serial No. 12,879,199 filed 10 September 2010.
  • TLR ligand-siRNA chimeric constructs can also be taken up by human monocytes, leading to gene silencing. See U.S. Patent Application Publication No. 2008/02144356, PCT International Publication No. WO 2008/094254, and U.S. Patent Application Serial No. 12,879,199 filed 10 September 2010.
  • CpG- Stat3 siRNA constructs are synthesized chemically without involvement of enzymes.
  • CpG-Stat3 siRNA chimeric molecule for inducing immune responses and antitumor effects, through blocking Stat3 in immune cells and/or in tumor cells, demonstrates a novel general approach: using TLR ligand oligonucleotides, which include CpG, polylrC (TLR3 ligand), polyG (TLR8 ligand), to deliver short RNA, which include both siRNA and activating RNA, to desired cells in vitro and in vivo, to stimulate innate immunity, to negate undesired effects and/or elicit desired effects through siRNA and/or activating RNA.
  • TLR ligand oligonucleotides which include CpG, polylrC (TLR3 ligand), polyG (TLR8 ligand)
  • creating chimeric molecule consisting of TLR ligand and siRNA and/or activating RNA, has great versatility and can be easily adapted for various gene targets. It also has flexibility: similar design can be adapted for different cell types capable of ODN/ORN uptake and internalization. Using a linker, modification of such approach to include multiple active moieties, such as multiple siRNA, with TLR ligand as a single agent for treating cancer and infectious disease is feasible. This approach can also be modified to enable small molecule drug delivery.
  • the present invention relates to specific chimeric molecules that are useful for the treatment of diseases.
  • the present invention provides a standard conjugate for use in preclinical and phase I studies.
  • the chimeric molecule comprises the components:
  • the present invention provides a chimeric molecule that is prepared to include modification sites for the sense strand sequence to produce a conjugate with increased serum stability.
  • the modified sense strand comprises: A. Human STAT3 SS siRNA (sense strand; underlined are deoxyribonucleotides; bold are chemically modified for increased nuclease resistance, e.g. 2'F-, 2'OMe-, LNA, nucleotides or other modifications described herein):
  • this sense strand is combined with the human CpG(D19)-STAT3 AS siRNA described above.
  • the present invention provides an alternative three-component conjugate with complementary "sticky ends" instead of fixed propanediol linker between CpG and siRNA moieties (to simplify synthesis).
  • the chimeric molecule comprises the components:
  • RNA Interference RNA Interference
  • RNAi The Nuts & Bolts ofsiRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.
  • Established B16 tumors (day 10 post 10 6 tumor cell challenge, >10 mm diameter) were treated with a single peritumoral injection of 5 ⁇ g CpG1668-oligonucleotide.
  • CpG-ODN treatment did not show significant antitumor activity in control mice (Stat3+/+) with heavy tumor load, the same treatment resulted in eradication of large B16 tumors (some of them reaching 1.5 cm in diameter) in mice lacking intact alleles within 3 days after injection.
  • the number of IFN- ⁇ - secreting T cells was strongly enhanced by CpG-treatment in the tumor-draining lymph nodes of Stat3-/- mice, as indicated by ELISPOT assay following ex vivo exposure to B16 tumor- specific l5E antigen.
  • CpG and Stat3 siRNA The 20 bp long single-stranded CpG1668 ODN sequence was fused to a double-stranded 25/27-mer Stat3siRNA.
  • the selection of optimized 25/27 Stat3siRNA is based on the report that Dicer-processed siRNA has enhanced silencing effects of target genes (Kim et al., 2005). In vitro cleavage assay confirmed that the chimeric
  • CpG-Stat3 siRNA construct is processed by recombinant Dicer enzyme, just like the 25/27-mer
  • CpG- Stat3-siR A uptake by confocal microscopy indicated rapid internalization of the chimeric construct with kinetics similar to the one previously reported for the CpG-ODN alone (Latz et al., 2004).
  • stable DC cell line DC2.4
  • the CpG-Stat3-siR A can be detectable as early as 15 min, with high uptake after 1 h of incubation.
  • CpG-Stat3-siRNA construct was found to colocalize with TLR9 within perinuclear endocytic vesicles.
  • CpG-Stat3-siRNA The ability of CpG-Stat3-siRNA to inhibit metastatic tumor growth was further demonstrated in the model of established B16 lung metastasis.
  • Systemic injection of 0.78 nmole CpG-Stat3-siR A led to significant reduction in the number of lung metastasis with lesser effect of CpG-scrambled-RNA and CpG- ODN alone, which is accompanied by upregulation of MHC class II, CD80 and CD40 molecules on tumor infiltrating DCs.
  • the ratio of effector to negative regulatory T cells within tumor microenvironment is considered an important indicator of the effect of adaptive immune responses against tumor.
  • TRP2 tumor antigen
  • the numbers of tumor-infiltrating NK cells and neutrophils are higher in mice treated with CpG-Stat3-siRNA.
  • TLR9 as well as the ability to take up CpG-based oligonucleotides is reportedly restricted to relatively rare population of plasmacytoid DCs in humans.
  • moderate levels of TLR9 expression have recently been found also in more common monocyte- derived DCs (MoDCs) isolated or expanded from peripheral blood mononuclear cells (PBMCs).
  • MoDCs monocyte- derived DCs isolated or expanded from peripheral blood mononuclear cells
  • We created an analogue chimeric oligonucelotide by fusion of CpG(D19) sequence optimized for activation of human TLR9-positive cells with the STAT3 -specific siRNA selected for the highest silencing effect in human cells.
  • CpG(D19)-STAT3-siRNA was incubated with human PBMCs to determine the level and specificity of oligonucleotide uptake.
  • Flow cytometric analysis revealed the internalization of fusion oligonucleotide by CD14+ monocytes but not by other PBMCs including CD3+ lymphocytes.
  • CpG(D19)-STAT3-siRNA uptake is detectable after short incubation time.
  • Chimeric oligonucleotide internalization is dose dependent within the range of 20 to 500 nM, with maximal near 100% uptake at the highest concentration after 24 h. Under these conditions, CpG(D19)-STAT3-siRNA reduced STAT3 expression by almost 75% comparing to CpG- scrambled-RNA control as measured by real-time PCR analysis.
  • Stat3 is activated in immune cells in the tumor microenvironment, promoting tumor immunosuppression
  • Stat3 is constitutively activated in tumor cells of diverse origin (Yu and Jove, 2004; Yu et al., 2007).
  • Stat3 activity intrinsic to the tumor cells upregulate a large range of genes critical for tumor growth, survival, angiogenesis/metastasis and immunosuppression. It is therefore highly desirable for any Stat3 inhibitor to be able to block Stat3 in the tumor cells.
  • CpG-Stat3 siRNA allows for siRNA delivery into various types of human B lymphoma cells, in a dose-dependent manner.
  • CpG-Stat3 siRNA can lead to Stat3 silencing in MPC 11 cells treated with 100 nM CpG-Stat3 siRNA for 24 h, as measured by real-time PCR.
  • MCP11 cells treated with CpG-Stat3 siRNA leads to accumulation in the G 2 M phase of cell cycle as measured by flow cytometry after propidium iodide staining.
  • CpG-Stat3 siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of diverse tumor-associated immune cells, and ultimately potent anti-tumor immune responses.
  • the findings described herein demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.
  • TLR9 oligonucleotide-binding TLRs
  • CpG ODN unmethylated CpG-motif
  • CpG ODN are efficiently internalized by various antigen-presenting cells, such as dendritic cells, macrophages and B cells, and their binding to TLR9 can initiate a cascade of innate and adaptive immune responses (Klimman et al., 2004; Barchet et al., 2008; Krieg, 2008). These immune cells are also critical components of the tumor microenvironment that actively promote oncogenesis (Kujawski et al., 2008; Yu et al., 2007; Kortylewski et al, 2008; Bollrath et al., 2009; Grivennikov et al., 2009).
  • CpG1688 alone or CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resulted in a similar increase in expression of co-stimulatory CD40 and CD80 molecules, suggesting that CpG-Stat3 siRNA retains its capacity to activate TLR9.
  • the immunostimulatory properties of CpG-siRNA conjugates do not differ from the effect of CpG alone as measured by production of inflammatory cytokines in primary cells and NF-KB/AP1 activation in a stable macrophage cell line designed for such test.
  • Stat3 siRNA and unconjugated Stat3 siRNA were labeled with fluorescein. Fluorescein-positive
  • DCs, macrophages, B cells, granulocytes and T cells were assessed by FACS analysis. Results from the flow cytometric analyses indicated that the chimeric CpG-Stat3 siRNA was efficiently taken up by both plasmacytoid (CD1 lc + B220 + ) and conventional (CDl lc + B220 ⁇ ) splenic DCs, macrophages (F4/80 + Grl ⁇ ) and B cells (B220 + CDl lc _ ), whereas uptake by splenic granulocytes (Grl + F4/80 ⁇ ) or T cells (CD3 + ) was minimal.
  • This uptake pattern reflects the known distribution of TLR9 expression in murine leukocyte subsets (Hemmi et al., 2000; Iwasaki and Medzhitov, 2004).
  • CDl lc + DCs were confirmed to express TLR9, as shown by intracellular staining of TLR9 in fixed splenocytes by flow cytometry.
  • Unconjugated Stat 3 siR A was not efficiently incorporated into DCs even after 24 h incubation, demonstrating that linkage to the TLR9 ligand facilitates siRNA uptake.
  • GpC-conjugated Stat3 siRNA which binds but does not activate TLR9 (Latz et al. 2004), failed to silence Stat3, suggesting a possible requirement of TLR9 activation for the CpG-siRNA to be further processed.
  • ESA electrophoretic mobility shift assays
  • C57BL/6 mice with aggressive poorly immunogenic B16 tumors (6-10 mm in diameter) were injected peritumorally with FITC-labeled CpG-Stat3 siRNA at 0.78 nmol (20 ⁇ g)/injection.
  • numerous myeloid cells accumulated at the site of CpG-Stat3 siRNA injection already 1 h later.
  • intravital two-photon microscopy indicated the presence of FITC-positive cells in tumor-draining lymph node, as early as 1 h after injection of the labeled construct, but not in the contralateral lymph nodes.
  • Peritumoral injections of the CpG-Stat3 siRNA resulted in relatively effective gene silencing in dendritic cells, macrophages and B cells accumulated in tumor draining lymph nodes, compared to control CpG-L c siRNA, as measured by quantitative real-time PCR.
  • Stat3 inactivation in CDl lc + dendritic cells isolated from tumor draining lymph nodes was confirmed at protein level.
  • quantitative real-time PCR and Western blotting indicate Stat3 silencing in the total tumor draining lymph nodes as well.
  • CpG-Luc siRNA conjugate was also used to confirm that CpG-siRNA conjugates are able to reduce protein expression specifically within myeloid cells in vivo.
  • mice over-expressing firefly luciferase under control of the ⁇ -actin promoter were challenged with tumor cells, followed by repeated peritumoral injections of CpG-Luc siRNA. Results from these experiments indicated effective inhibition of luciferase activity in CDl lb + myeloid cells but not in CD4 + lymphocytes within tumor-draining lymph nodes.
  • CpG and LPS treatment activates Stat3 (Samarasinghe et al., 2006; Kortylewski et al., 2009b; Benkhart et al., 2000), which acts as a negative feedback mechanism to constrain Thl immune responses. Therefore, we assessed whether the CpG-Sto/i-siRNA conjugates could reverse the immunosuppressive effects imposed by the tumor-microenvironment and at the same time allow effective antitumor immunity induced by TLR9 triggering.
  • Local treatment with CpG-Stat3 siRNA oligonucleotides inhibited growth of subcutaneously growing B16 melanoma (3-5 mm in diameter at the initial treatment).
  • CpG-Stat3 siRNA oligonucleotides inhibited growth of both a poorly immunogenic variant of K1735 melanoma, C4 (Xie et al., 2004), and CT26 colon carcinomas in C3H and BALB/c mice, respectively. Furthermore, CpG-Stat3 siRNA treatment of the carcinoembryonic antigen (CEA) transgenic C57BL/6 mice bearing MC38 colon carcinomas expressing CEA led to tumor regression.
  • CEA carcinoembryonic antigen
  • CpG-Stat3 siRNA intravenous injections of CpG-Stat3 siRNA can lead to gene silencing and antitumor effects.
  • Intravenous injection of CpG-Stat3 siRNA (0.78 nmol) reduced Stat3 expression in dendritic cells within tumor-draining lymph nodes relative to CpG-scrambled RNA.
  • CpG- Stat3 siRNA was also tested the ability of systemic delivery of CpG- Stat3 siRNA to inhibit metastatic tumor growth in an established B16 lung metastasis model. Mice with disseminated B16 tumor cells were treated systemically with CpG-»3 ⁇ 4ar3 siRNA thrice weekly for two weeks.
  • the ratio of effector to regulatory T cells within the tumor microenvironment is considered to correlate well with the effect of adaptive immune responses on tumor progression and metastasis (Bui et al., 2006).
  • ELISPOT assays to determine IFNy production by T cells isolated from tumor draining lymph nodes in response to recall stimulation with TRP2 peptide indicated that in vivo CpG-Stat3 siRNA administration indeed induced antigen-specific CD8 + T cells.
  • TLR oligonucleotide agonists to siRNAs.
  • These conjugates encompass three activities in a single molecule: targeting to immune cells, which include DCs, macrophages, and B cells, TLR activation and immune checkpoint silencing.
  • immune cells which include DCs, macrophages, and B cells
  • TLR activation and immune checkpoint silencing In addition to TLR9, several other intracellular TLRs, such as TLR3, TLR7 and TLR8 also recognize nucleic acids, suggesting a broad application of this approach using various ligands for these TLRs to deliver various siRNAs into different immune cells.
  • TLRs are important for stimulating dendritic cell maturation, antigen uptake and presentation, leading to CTL activation and CD4 + T helper cell differentiation.
  • TLR agonist-siRNA approaches can further stimulate desired immune responses for treating diseases such as cancer and infections.
  • binding to TLR9 is necessary for CpG-mediated immune activation, it remains to be fully explored how CpG ODN enter cells (Latz et al, 2004).
  • TLR9 is required for CpG-siRNA mediated gene silencing. While the exact underlying mechanism(s) remains to be determined, it is possible that triggering TLR9 could effect either endosomal release of CpG-siRNA into the cytoplasm, or/and its intracellular processing.
  • TLR9 is expressed in different types of mouse dendritic cells, its expression is more selective in humans. While the highest levels of constitutive TLR9 expression is observed on human plasmacytoid DCs and B cells, it is also expressed at lower levels on human monocytes and macrophages (Iwasaki and Medzihitov, 2004). These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity (Kanzler et al., 2007; Krieg, 2008; Marshner et al., 2005; Klinman et al., 2008).
  • Stat3 and several other molecules produced by the tumor myeloid population, and possibly tumor- associated B cells are critical for tumor immunosuppression (Yu et al., 2007), and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival (Kujawski et al., 2008; Bollrath et al., 2009; Grivennikov et al., 2009; Lee et al., 2009).
  • other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies.
  • the siRNA in the chimeric construct is unmodified and negatively charged. Chemically modifying the siRNA to prolong serum stability and to neutralize the negative charge of the siRNA to facilitate endosomal release may improve the efficacy of TLR agonist-siRNA approach.
  • Our results show the use of oligonucleotide TLR agonists for siRNA delivery into tumor-associated myeloid cells and B cells to inhibit expression of tumor-promoting/immunosuppressive molecules while activating TLR(s) for immune activation.
  • CpG(A)-STAT3 siRNA i.e., CpG(Ol9)-STAT3 siRNA
  • CpG(Ol9)-STAT3 siRNA can target human TLR9 + cells in vivo.
  • the intravenously injected CpG(A)-S73 ⁇ 4 T3 siRNA led to STAT 3 gene silencing in human MV4- 11 acute myeloid leukemia (AML) cells residing in the bone-marrow of immunodeficient NOD/SCID IL-2RY nuI1 (NSG) mice (Fig. 1A).
  • CpG(D19)-human STAT3 siRNA antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker
  • CpG(D19)-Luciferase siRNA antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker
  • Luciferase siRNA sense strand ; underlined are deoxyribonucleotides
  • CpG(7909)-human STAT3 siRNA antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker
  • CpG(7909)-Lucif erase siRNA (antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker)
  • CpG-siRNAs were used for normal human immune cells. Multiplex assays indicated that CpG(O ⁇ 9)-STAT3 siRNA induced more desirable cytokine expression profile comparing to the class B-based CpG(7909)-STAT3 siRNA and class C-based CpG(2429)-STAT3 siRNA (Fig. 3).
  • CpG(2429)-human STAT3 siRNA antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker
  • CpG(2429)-Luciferase siRNA (antisense strand; underlined are deoxyribonucleotides, asterisks indicate phosphothioated sites, X indicates single C3 carbon chain linker) 5' T*C*G i
  • Alexopoulou L. et al. (2001). Recognition of double-stranded RNA and activation of NK-KB by tollOlike receptor 3. Nature 413:732-738.
  • TRBP a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 6:961-967.

Abstract

L'invention concerne des méthodes et des compositions pour améliorer la fonction immunitaire et traiter des maladies, notamment le cancer, les maladies infectieuses et les maladies auto-immunes. L'invention concerne plus particulièrement des molécules multifonctionnelles pouvant être administrées à des cellules d'intérêt afin de bloquer STAT3 pour traiter des maladies et améliorer la fonction immunitaire.
PCT/US2011/051042 2011-03-22 2011-09-09 Méthodes et compositions pour traiter le cancer ou d'autres maladies WO2012128785A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161466086P 2011-03-22 2011-03-22
US61/466,086 2011-03-22

Publications (1)

Publication Number Publication Date
WO2012128785A1 true WO2012128785A1 (fr) 2012-09-27

Family

ID=46879659

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/051042 WO2012128785A1 (fr) 2011-03-22 2011-09-09 Méthodes et compositions pour traiter le cancer ou d'autres maladies

Country Status (1)

Country Link
WO (1) WO2012128785A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107980001A (zh) * 2015-07-02 2018-05-01 希望之城 包括硫代磷酸化寡脱氧核苷酸的化合物和组合物及其使用方法
WO2019048631A1 (fr) 2017-09-08 2019-03-14 Mina Therapeutics Limited Compositions de petits arn activateurs de hnf4a et procédés d'utilisation
US10253318B2 (en) 2007-01-26 2019-04-09 City Of Hope Methods and compositions for the treatment of cancer or other diseases
US10538761B2 (en) 2014-01-13 2020-01-21 City Of Hope Multivalent oligonucleotide assemblies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030060440A1 (en) * 1999-04-12 2003-03-27 Dennis Klinman Oligodeoxynucleotide and its use to induce an immune response
US20080214436A1 (en) * 2007-01-26 2008-09-04 City Of Hope Methods and compositions for the treatment of cancer or other diseases
US20100298409A1 (en) * 2007-09-17 2010-11-25 Intradigm Corporation Compositions comprising stat3 sirna and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030060440A1 (en) * 1999-04-12 2003-03-27 Dennis Klinman Oligodeoxynucleotide and its use to induce an immune response
US20080214436A1 (en) * 2007-01-26 2008-09-04 City Of Hope Methods and compositions for the treatment of cancer or other diseases
US20100298409A1 (en) * 2007-09-17 2010-11-25 Intradigm Corporation Compositions comprising stat3 sirna and methods of use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHIU ET AL.: "siRNA function in RNAi: A chemical modification analysis.", RNA., vol. 9, no. 9, 2003, pages 1034 - 1048 *
HOENE ET AL.: "Human monocyte-derived dendritic cells express TLR9 and react directly to the CpG-A oligonucleotide D19.", J LEUKOC BIOL., vol. 80, no. 6, 2006, pages 1328 - 1336 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10253318B2 (en) 2007-01-26 2019-04-09 City Of Hope Methods and compositions for the treatment of cancer or other diseases
US11208654B2 (en) 2007-01-26 2021-12-28 City Of Hope Methods and compositions for the treatment of cancer or other diseases
US10538761B2 (en) 2014-01-13 2020-01-21 City Of Hope Multivalent oligonucleotide assemblies
US11535847B2 (en) 2014-01-13 2022-12-27 City Of Hope Multivalent oligonucleotide assemblies
CN107980001A (zh) * 2015-07-02 2018-05-01 希望之城 包括硫代磷酸化寡脱氧核苷酸的化合物和组合物及其使用方法
US10758624B2 (en) 2015-07-02 2020-09-01 City Of Hope Compounds and compositions including phosphorothioated oligodeoxynucleotide, and methods of use thereof
US11464865B2 (en) 2015-07-02 2022-10-11 City Of Hope Compounds and compositions including phosphorothioated oligodeoxynucleotide, and methods of use thereof
WO2019048631A1 (fr) 2017-09-08 2019-03-14 Mina Therapeutics Limited Compositions de petits arn activateurs de hnf4a et procédés d'utilisation
EP4233880A2 (fr) 2017-09-08 2023-08-30 MiNA Therapeutics Limited Compositions de petits arn activateurs de hnf4a et procédés d'utilisation

Similar Documents

Publication Publication Date Title
US11208654B2 (en) Methods and compositions for the treatment of cancer or other diseases
US20080214436A1 (en) Methods and compositions for the treatment of cancer or other diseases
US10041071B2 (en) Cell-type specific aptamer-siRNA delivery system for HIV-1 therapy
JP5637849B2 (ja) ２本鎖ポリヌクレオチド
JP6453212B2 (ja) キラル制御
DK2341943T3 (en) COMPOSITIONS AND METHODS OF SPECIFIC INHIBITION OF DSRNA REPRINT WITH MODIFICATIONS
US20180208927A1 (en) Multi-targeted single entity conjugates
WO2018003739A1 (fr) Composite d'acide nucléique comprenant un ligand fonctionnel
KR20100106314A (ko) Ｒｎａ 간섭 효과가 높은 지질 수식 2중쇄 ｒｎａ
JP2024501857A (ja) 環状ジスルフィド修飾リン酸ベースのオリゴヌクレオチドプロドラッグ
WO2012128785A1 (fr) Méthodes et compositions pour traiter le cancer ou d'autres maladies
Kim et al. Immune activation by siRNA/liposome complexes in mice is sequence-independent: lack of a role for Toll-like receptor 3 signaling
US20110071210A1 (en) Methods and compositions for the treatment of cancer or other diseases
KR20080044909A (ko) 세포 내로 리보핵산의 전달을 위한 약학 조성물
US20240124875A1 (en) Rnai conjugates and uses thereof
Akhtar Pharmacological reprogramming of macrophages through aptamiRs
CN117625610A (zh) 用于增强STING表达的saRNA、缀合物及药物组合物
KR20200133681A (ko) PD-1의 발현을 억제하는 비대칭 siRNA
MX2008003380A (en) Pharmaceutical compositions for delivery of ribonucleic acid to a cell
MX2007003667A (en) Method of treating an inflammatory disease by double stranded ribonucleic acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11861601

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11861601

Country of ref document: EP

Kind code of ref document: A1